The long-term goal of our research has been to understand how cytoplasmic protein-tyrosine kinases regulate the growth properties of immune cells in response to extracellular stimuli. Our current focus is on the tyrosine kinase, Syk, which was discovered as part of this work. Through its substrates and binding partners, Syk couples the B cell receptor for antigen (BCR) to multiple intracellular signaling pathways resulting in one of several possible physiological outcomes that can vary from proliferation to apoptosis depending on the repertoire of downstream signals that are sent. Syk has functions not easily explained by its known activities and its known substrates at the site of the clustered BCR complex including an ability to suppress cellular responses to apoptotic stimuli. This function has made Syk an attractive target for anti-cancer therapeutics. The mechanism by which Syk dissociates from the BCR complex and is subsequently targeted to other cellular docking sites will be investigated as will the nature of these alternative binding proteins. The putative substrates that Syk phosphorylates as a consequence of its re-localization, as identified by sophisticated mass spectrometric analyses, will be characterized and the consequences of their phosphorylation analyzed to investigate new pathways that are regulated directly by Syk. These studies will be extended to primary B cells using cells derived from a new mouse model in which the activity of Syk can be unambiguously inhibited using a powerful chemical genetics approach. We plan to accomplish three specific aims: 1) to characterize the role of tyrosine-130 phosphorylation in the modulation of Syk-receptor and Syk-protein interactions, 2) to characterize novel Syk substrates and signaling pathways identified through proteomic and phosphoproteomic screens, and 3) to characterize the role of Syk in signaling in primary B cells through chemical genetics. Methodologies to be employed include 1) biochemical and physical evaluations of protein-peptide and protein- protein interactions, 2) proteomic and phosphoproteomic analyses of interacting proteins and kinase substrates, 3) molecular and cellular studies on protein function and 4) the use of chemical inhibitors to block kinase function in cells derived from genetically manipulated mice expressing an engineered kinase.